Different functional types of nicotinic acetylcholine receptors (AChRs) are expressed during the development of skeletal muscle. The molecular mechanisms underlying expression of the different AChR channel types will be investigated in Xenopus laevis. The AChR is an assembly of diverse subunits, and the functional properties of the receptor depend on its subunit composition. To understand AChR development, we must know how expression of the different subunits is regulated during development and how the various subunits affect receptor function. We propose to investigate the pathway leading to AChR expression with respect to the following: (1) inductive extracellular signals, (2) master regulatory genes, (3) AChR subunit transcript levels, and (4) subunit composition of functional receptors. The first step in this pathway involves the role of extracellular signals in initiating or modulating AChR expression. Our studies will focus on peptides, particularly transforming growth factors and calcitonin gene-related peptide, which are likely to be important in regulating AChRs. Their effects on AChR function will be studied in organ- cultured, developing muscle. Secondly, there is evidence suggesting that transforming growth factor (TGF)-beta2 may activate AChR genes via MyoD, a master regulatory gene involved in muscle differentiation. We will determine if TGF-beta2 induces expression of MyoD and if this is necessary for activation of AChR genes in response to the peptide. Thirdly, since different types of muscle express predominantly different classes of AChRs, levels of the different subunit transcripts will be compared in these muscles to see if transcript availability determines the types of receptors expressed. Finally, the effect of subunit composition on receptor function will be studied. We will examine the functional properties of receptors with different subunit compositions expressed in oocytes, in developing muscle, and in stably transfected cells. In these experiments, receptor function will be measured by RNase protection analysis. Complementary DNAs for all of the known AChR subunits except beta and epsilon have been cloned and are available for this study. During the first year of the project, the cloning of the beta and epsilon subunit cDNAs will be undertaken in collaboration with Dr. Gail Mandel and Dr. Paul Brehm at SUNY, Stony Brook.